Ocular itch is a refractory symptom of many ocular conditions, and severely affect the quality of life and productivity. Ocular itch is thought to be mediated by a group of primary sensory neurons residing in the trigeminal ganglia. These neurons detect endogenous itch-inducing mediators (pruritogens) via their peripheral axons in the conjunctiva, and transmit signals to the brainstem via their central axons. However, the molecular identification of these itch-sensing neurons remains elusive. Previously, we identified a novel itch receptor, called MrgprA3. We found that MrgprA3 marks a highly restricted population of primary sensory neurons that mediates acute and chronic itch in the skin. Interestingly, our latest results revealed that MrgprA3-expressing sensory neurons also project to the conjunctiva but not to other mucosal membrane tissues examined. However, the function of MrgprA3-expressing neurons in ocular itch remains to be determined. This proposal aims to uncover the neural mechanisms of ocular itch. Aim 1 will characterize the innervation pattern and physiological properties of MrgprA3-expressing sensory fibers in the conjunctiva. Using genetic labeling tools, we will perform detailed anatomical analysis of the innervation of MrgprA3-expressing sensory fibers in the conjunctiva during development and in adulthood. In addition, we will test whether MrgprA3-expressing sensory fibers in the conjunctiva can be activated by various pruritogens. These studies will provide important information about the potential role of MrgprA3-expressing sensory fibers in ocular itch. Aim 2 will investigate whether MrgprA3-expressing neurons mediate acute ocular itch. We will determine whether ablation of MrgprA3-expressing neurons alleviates the ocular itch produced by various pruritogen. Furthermore, we will examine the behavioral consequence of selective activation of MrgprA3-expressing sensory fibers in the conjunctiva. These loss-of-function and gain-of-function studies will firmly establish the role of MrgprA3- expressing neurons in ocular itch, which will, for the frst time, unravel the neural mechanism of ocular itch at the peripheral level. In Aim 3, we seek to understand the interaction between MrgprA3-expressing sensory fibers and mast cells in allergic conjunctivitis and determine whether MrgprA3-expressing fibers mediate related ocular itch. Based on our preliminary data, we hypothesize that mast cells release endogenous pruritogens upon allergen-induced degranulation and excite MrgprA3-expressing sensory fibers to induce itch. Using a novel imaging tool combined with molecular and behavioral analysis, we will investigate this hypothesis. These studies will reveal the neural basis underlying ocular itch that occurs in perennial and seasonal allergic conjunctivitis and will have a significant impact on both the study of ocular itch pathogenesis and the clinical treatment of chronic itch.